Determination of acid phosphatase activity in normal human lymphocytes.

Acid phosphatase levels in pure lymphocytes from normal individuals were determined cytochemically on intact cells and spectrophotometrically on cell extracts. Good correlation was demonstrated between the results obtained with both methods. Biochemical assay showed that a normal range of 7.2 +/- 0.9 and 7.1 +/- 1.0 mMoles per 10(6) lymphocytes was obtained without and with tartrate inhibition, respectively. A significant loss of acid phosphatase level in the extract was found on storage. Seventy-five, 49 and 45 percent of the acid phosphatase activity remained after 3.5, 24, and 48 hours refrigeration at 4 degrees C, with 51 percent and 32 percent after 24 and 48 hours refrigeration at -20 degrees C, respectively. The spectrophotometric assay is less variable than the cytochemical stain. The widely utilized cytochemical method, although less reproducible, allows for evaluation of intracellular distribution of the enzyme in addition to level of activity.

Determination of plasminogen in human serum or plasma by a fibrometric method.

A simple, fast, reproducible, and reliable fibrometer method for determination of plasma or serum plasminogen was developed. Results obtained with this method were compared to those obtained with a Coseinolytic method and a Chromogenic Substrate S-2251 method. For all three procedures, serum or plasma samples were acidified before the activation process. Conversion of plasminogen to plasmin during a controlled incubation time at 37 degrees C was accomplished with urokinase, and/or streptokinase. Precision which was determined on multiple aliquoted serum samples showed that the within day coefficient of variations were 4.35 percent and 4.82 percent, and an overall coefficient of variation of 7.68 percent. Satisfactory correlation was also demonstrated on 26 patients' plasmas. A correlation of 0.90 and 0.88 was found between the Caseinolytic assay and the Chromogenic assay, and the clotting time assay. A fibrometric normal range of 68 to 150 percent activity was obtained on 24 normal serum samples.

Kinetic determination of granulocyte alkaline phosphatase by the GEMSAEC.

Granulocyte alkaline phosphatase (ALP) activity is measured kinetically using the GEMSAEC centrifugal analyzer and p-nitrophenylphosphate as substrate. Measurements of enzyme activity are made at pH 9.8 utilizing 1.0 M diethanolamine as a buffer containing 0.5 mM magnesium chloride. Granulocytes are separated from heparinized blood using dextran sedimentation, followed with ficoll-hypaque separation. The separated cells are suspended in saline, and the red cells are lysed. Manual counting of stained smears of the cells isolated showed that 95 percent of these cells were granulocytes. After lysing the red cells, the granulocyte suspension is rinsed in 12.5 percent heat inactivated serum in saline and separated by centrifugation. After 200 microliters of the diluent solution (12.5 percent heat inactivated serum in saline) is added to the cells, they are sonicated for seven sec at 0 degrees in an ice bath. The extracts are then assayed kinetically for ALP activity. Granulocyte ALP activity varies from day to day in normal individuals. The daily variation of ALP activity observed correlates closely with and is confirmed by the Sigma Histochemical Procedure based on Kaplow' Scoring Method. The coefficient of variation of the ALP method within assay performed on two consecutive days on a pooled sample is 0.75 percent and 1.38 percent, respectively, and between assays 4.82 percent.